Sodium bicarbonate cotransporters in the kidney contribute to the maintenance of whole body acid-base homeostasis. These integral membrane proteins play an important role in regulating intracellular pH and in mediating transepithelial sodium and bicarbonate tranport. Our understanding of the functional characteristics of these transporters has improved following the recent cloning of several members of the sodium bicarbonate cotranport (NBC) family. We have recently cloned and functionally characterized NBC3, a unique member of the NBC family which is stilbene-insensitive, electroneutral, and EIPA-inhibitable. In kidney, NBC3 is sorted to the apical membrane in type A intercalated cells and to the basolateral membrane in type B intercalated cells. Furthermore, NBC3 co-localized and co-immunoprecipitated with the vacuolar H+-ATPase have consensus PDZ-interacting domain, -ETAL and -DTAL respectively, which could play an important role in membrane sorting. On this basis, we screened a human kidney cDNA library in a yeast two-hybrid assay using the C-terminus of NBC3 as bait, and isolated a novel protein, NBC3BP, which has three potential PDZ-binding domains. NBC3BP was localized to the apical membrane of type A intercalated cells and was co-immunoprecipitated with NBC3 from kidney. We hypothesize that NBC3 and 58 kD subunit of the vacuolar H+-ATPase are sorted to the apical membrane of type A intercalated cells, via interaction of their C-terminal PDZ-interacting domains with the PDZ-binding protein, NBC3BP, which is expressed on the apical membrane of this cell type and not in type B intercalated cells. We will use the MDCK I cell line which sorts NBC3BP and NBC3 to the apical membrane similar to the type A intercalated cell as a model system for achieving the goals of this proposal. Successful completion of this project will enhance our understanding of the molecular mechanisms responsible for determining the polarity of acid-base transporters.